JP3801937B2 - Modified metallothionein - Google Patents

Description

【００４３】
試験例２：pH変化に対する安定性の測定
実施例３で作製した金属結合メタロチオネインおよびβドメインペプチドを20 mM Tris-HCl緩衝液（pH 7.5）で3 nmol/mlに希釈した後、HClでpH 2.5に調製して金属を解離させた。次いで、Tris溶液を添加してpHを徐々に上昇させ、メタロチオネインおよびβドメインペプチドの安定性をUV吸収の変化として測定した。なお、カドミウム結合のものについては250nm、亜鉛結合のものは220nmの吸光度を測定した。
【００４４】
結果は図１および図２に示したとおりである。図１は、野生型および変異型のカドミウム結合メタロチオネインおよびβドメインペプチドのpH滴定曲線であり、図２は亜鉛結合メタロチオネインおよびβドメインペプチドのpH滴定曲線である。いずれの金属を結合した場合も、野生型の全長メタロチオネインおよびそのβドメインペプチドに比較し、２箇所のアミノ酸置換を有するメタロチオネイン変異体およびβドメインペプチド変異体は低pHで立ち上がり、その勾配も急であることから、pH安定性が向上し、金属結合能が増強されていることが確認された。
【００４５】
【発明の効果】
以上詳しく説明したとおり、この出願の発明によって、野生型メタロチオネインに比べて金属結合能がさらに増強された改変型メタロチオネインが提供される。
【００４６】
【配列表】
<110> Japan Science and Technology Corporation
<120> A modified metallothionein
<130> NP02127-YS
<140>
<141>
<160> 1
<210> 1
<211> 61
<212> PRT
<213> Homo sapiens
<400> 1
Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala
50 55 60
【図面の簡単な説明】
【図１】野生型および変異型のカドミウム結合メタロチオネインおよびβドメインペプチドのpH滴定曲線である。pTIMTIは完全長野生型、pTIMTID11C/S28Cは完全長変異型、pTIMTIIはβドメイン野生型、pTIMTIID11C/S28Cはβドメイン変異型を示す。
【図２】野生型および変異型の亜鉛結合メタロチオネインおよびβドメインペプチドのpH滴定曲線である。pTIMTIは完全長野生型、pTIMTID11C/S28Cは完全長変異型、pTIMTIIはβドメイン野生型、pTIMTIID11C/S28Cはβドメイン変異型を示す。[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to a modified metallothionein. More specifically, the invention of this application relates to a modified metallothionein that has further enhanced the metal binding ability of wild-type metallothionein and is useful for the development of pharmaceuticals, diagnostic agents, etc., or for the recovery of heavy metals contaminating the environment. is there.
[0002]
[Prior art]
Metallothionein (MT) is a protein present in cells of yeast, mold, plant, invertebrate, vertebrate, etc., and is characterized by the following properties, for example.
(1) Bonds with various heavy metals (Cd, Zn, Cu, Hg, Ag, etc.) and has a high metal content per molecule.
(2) Low molecular weight protein. Without metal, it is about 3000 in N. crassa and 6000-7000 in many vertebrates.
(3) Contains many amino acid residues Cys. In mammalian metallothionein, 1/3 of all amino acid residues are Cys (for example, in the case of human metallothionein-II, 20 of 61 amino acids are Cys). Heavy metals are linked to metallothionein via the thiol group of Cys.
(4) Absence of absorbance at 280 nm because it does not contain aromatic amino acids. However, when cadmium is bound, there is absorption at around 254 nm due to the mercaptide bond between cadmium and the thiol group. This absorption disappears when cadmium is separated from metallothionein at low pH.
[0003]
MT is known to form two clusters from the results of ¹³ Cd NMR analysis. In the A cluster, 11 Cys are bonded to 4 atoms of cadmium or zinc, and it corresponds to the 31st-61st amino acid region of the C-terminal of metallothionein, and is also called an α domain. On the other hand, the B cluster has 9 Cys bonded to 3 atoms of cadmium or zinc, corresponds to the 1-30th amino acid region at the N-terminal of metallothionein, and is also called β domain. By X-ray analysis, the α and β domains are known to be spherical with a diameter of 1.5 to 2.0 nm.
[0004]
Various metals are bound to metallothionein isolated and purified from organs and tissues of organisms. For example, metallothionein purified from the liver of a human autopsy is known to be rich in zinc while the kidney is rich in cadmium. In addition, it is known that metallothionein containing a large amount of the metal can be obtained from animals, livers and kidneys experimentally administered with cadmium and zinc.
[0005]
Metallothionein refers to a state in which a metal is bound in vivo and in vitro, and a substance not bound to a metal is defined as “apometallothionein”. However, in the following description, a state in which a metal is bound and a state in which a metal is not bound are collectively referred to as “metallothionein”, and a purified protein that is not bound to a metal is particularly referred to as apometallothionein.
[0006]
At present, the biological functions of metallothionein are listed below. In other words, it has the ability to bind heavy metals, and its expression is controlled at the gene transcription level by heavy metals, oncogene products and stress stimuli, reducing the toxicity of heavy metals (Cd, Hg, etc.) Metabolism of essential metals (Cu, Zn, etc.) is considered. Alternatively, since it contains a large amount of Cys, it is assumed to be involved in the regulation of redox potential and sulfur metabolism. Furthermore, it is known that there is a high correlation between the nerve growth inhibitory factor deficient in the brain of Alzheimer's disease patients and the amount of metallothionein, and that metallothionein is also involved in anticancer drug resistance in cancer patients.
[0007]
Therefore, metallothionein is expected to play a role as a drug for maintaining homeostasis, a part of a drug component for treatment of Alzheimer's disease, cancer and the like, or a target protein for searching for these drug components.
[0008]
On the other hand, apart from the biological function of metallothionein, focusing on its excellent heavy metal binding ability, for example, the use of metallothionein for the purpose of monitoring and removing heavy metals that cause environmental pollutants has been studied. For example, Japanese Patent Application Laid-Open No. 5-180747 discloses a metallothionein-containing heavy metal monitor for the purpose of detecting heavy metals in water.
[0009]
As described above, the usefulness of metallothionein has been pointed out in the fields of medicine, pharmaceutical production, environmental purification, etc., but it is difficult to isolate and purify metallothionein in large quantities from living tissues. Various attempts have been made to produce metallothionein by the method. For example, the inventors of this application is the monkey metallothionein -II cDNA bound to P _R promoter and SD sequence downstream, already reported the expression of MT in the 14mg / g (total protein) in direct expression in E. coli ( Appl. Environ. Microbiol. 53: 204-207, 1987). Similarly, the inventors of this application conducted total synthesis of the human metallothionein-II gene with reference to the amino acid sequence of human metallothionein-II, the DNA sequence, and the design method of synthetic DNA for E. coli expression. It has also been found that fusion expression with β-galactosidase has succeeded in expressing about 24% of the total protein, and that the resulting metallothionein exhibits significant metal binding activity (J. Ferment Bioeng. 77 (2): 113-118, 1994). In addition, several examples of metallothionein expression by E. coli are known (for example, J. Biochem. 104: 924-926, 1988; Biochem. Biophys. Acta. 951: 230-234, 1988; J. Biotechnol. 8: 207-220, 1988; Gene 83: 95-103, 1989; Biochem. Biophys. Acta. 1048: 178-186, 1990).
[0010]
Furthermore, the inventors of this application have invented a metallothionein multimer in which a plurality of metallothionein molecules are linked and a method for producing the same in order to express recombinant metallothionein in a stable state and in large quantities. (JP 2000-60561 A).
[0011]
[Problems to be solved by the invention]
As described above, there is a certain target for mass production of metallothionein using genetic engineering techniques. However, considering that metallothionein is useful as a component of pharmaceuticals and diagnostic agents due to its metal-binding ability, or as a material for purifying heavy metal-contaminated environments, the metal-binding ability of metallothionein is further increased. Enhancement is also an important factor for further improving its usefulness. This is because such a metallothionein with enhanced metal binding ability is expected to exert a medical function in a smaller amount, and to expand the usage amount and application range for environmental purification.
[0012]
The invention of this application has been made in view of the circumstances as described above, and it is an object of the present invention to provide a modified metallothionein that further enhances its metal binding ability compared to wild-type metallothionein.
[0013]
Another object of the invention of the application is to provide a material for enabling genetic engineering production of such a modified metallothionein.
[0014]
Furthermore, an object of the invention of this application is to provide a method for screening a modified metallothionein with enhanced metal binding ability.
[0015]
[Means for Solving the Problems]
This application provides, as a first invention for solving the above-mentioned problems, a modified metallothionein in which one or more non-cysteine residues in the amino acid sequence of metallothionein are substituted with cysteine residues (Cys).
[0016]
In the modified metallothionein of the first invention, the metallothionein is a human metallothionein consisting of the amino acid sequence of SEQ ID NO: 1, and the 11th aspartic acid residue (Asp) and the 28th amino acid sequence of the amino acid sequence of SEQ ID NO: 1 A preferred embodiment is that the serine residue (Ser) is substituted with a cysteine residue (Cys).
[0017]
As a second invention, this application provides a polynucleotide encoding a modified metallothionein in which one or more non-cysteine residues in the amino acid sequence of metallothionein are substituted with cysteine residues (Cys).
[0018]
In the polynucleotide of the second invention, the metallothionein is a human metallothionein consisting of the amino acid sequence of SEQ ID NO: 1, and the 11th aspartic acid residue (Asp) and the 28th serine in the amino acid sequence of SEQ ID NO: 1. A preferred embodiment encodes a modified metallothionein in which the residue (Ser) is substituted with a cysteine residue (Cys).
[0019]
This application provides, as a third invention, a recombinant vector having the polynucleotide of the second invention.
[0020]
This application provides, as a fourth invention, a transformed cell using the recombinant vector of the third invention.
[0021]
This application provides, as a fifth invention, a modified metallothionein that is an expression product of the recombinant vector of the third invention or a product of the transformed cell of the fourth invention.
[0022]
Furthermore, this application provides, as a sixth invention, a method for screening a modified metallothionein with enhanced metal binding ability, comprising the following steps:
(a) preparing a plurality of metallothionein mutants in which one or more non-cysteine residues in the amino acid sequence of metallothionein are replaced with cysteine residues (Cys), with different types of substituted amino acid residues;
(b) contacting each metallothionein variant with one or more metals;
(c) selecting a metallothionein variant that binds more metal atoms than wild-type metallothionein and / or a metallothionein variant that is more stable against pH changes than wild-type metallothionein.
A modified metallothionein is characterized by comprising:
[0023]
Hereinafter, embodiments will be shown and each invention of this application will be described in detail.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
The modified metallothionein of the first invention is a polypeptide in which one or more non-cysteine residues in the amino acid sequence of wild-type metallothionein are substituted with cysteine residues (Cys).
[0025]
“Wild-type metallothionein” means natural metallothionein derived from various species, and those having known genes (cDNA) and amino acid sequences are particularly preferred. For example, the metallothionein gene is known in N. crassa , yeast, rainbow trout, monkey, human, etc., and its cDNA sequence and amino acid sequence can be easily known by those skilled in the art from an existing sequence database (GenBank, etc.). For example, four isoproteins are known for human metallothionein, including Genbank Accession No. X97261 for metallothionein-I, GenBank Accession No. X97260 for metallothionein-II, GenBank Accession No. XM # 008111 for metallothionein-III, etc. It has been.
[0026]
The “modified metallothionein” is a polypeptide whose metal binding ability is enhanced by substituting one or more non-Cys residues with a Cys residue in the amino acid sequence constituting the wild-type metallothionein. The modified metallothionein may be the same size as the wild-type metallothionein, or a part of the wild-type metallothionein (for example, the β domain or the The peptide may be an α domain or a peptide having 10 amino acid residues or more partially including both.
[0027]
Furthermore, the Cys residue to be substituted with the non-Cys residue may be a natural Cys residue or any Cys residue derivative provided that the binding ability to metal is good. Good. Such Cys residue derivatives include, for example, cysteinyl groups such as bromo-trifluoroacetic acid, α-bromo-β- (5-imidazolyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2- Pyridyl disulfide; methyl 2-pyridyl sulfide; p-chloromercuribenzoic acid; 2-chloromercuri-4 nitrophenol; or chloro-7-nitrobenzo-oxa-1,3-diazole .
[0028]
One method for substituting one or more non-Cys residues in the amino acid sequence of wild-type metallothionein with a Cys residue is a method for producing a polypeptide containing a specific base substitution by a known peptide synthesis method. Peptide synthesis can be performed using various solid phase methods (see, for example, Science 269: 202, 1995; Methods Enzymol. 289: 3-13, 1997), and for example, using ABI431A peptide synthesizer (Perkin Elmer) It can also be done automatically by using it.
[0029]
Another method for substituting one or more non-Cys residues in the amino acid sequence of wild-type metallothionein with a Cys residue is to prepare a polynucleotide (second invention) encoding an amino acid sequence with base substitution. This is a method for producing a modified metallothionein as a culture of transformed cells (fourth invention) by recombining a polynucleotide with an expression vector (third invention).
[0030]
The polynucleotide of the second invention comprises three nucleotides (triplet) encoding non-Cys residues in a nucleic acid sequence (genomic DNA, mRNA, and cDNA synthesized from mRNA) encoding a known wild-type metallothionein. It can be made by substituting a triplet (aga, agc) encoding the residue. The triplet substitution can be performed by a known method such as a method using a mutation kit or the like, or a mutagenesis PCR method. It can also be chemically synthesized by a known polynucleotide synthesis method (for example, Nucleic Acid Res. 25: 3440-3444, 1997).
[0031]
By using the polynucleotide of the second invention, a modified metallothionein (the fifth invention) can be produced from the recombinant vector of the third invention and / or the transformed cell of the fourth invention. For example, if a polynucleotide is recombined into an expression vector having an RNA polymerase promoter and the recombinant vector is added to an in vitro translation system such as a rabbit reticulocyte lysate or wheat germ extract containing an RNA polymerase corresponding to the promoter, the modification Type metallothionein can be produced in vitro. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Examples of vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T718, pT7 / 319, and pBluescript II. If the polynucleotide is expressed in an appropriate host-vector system, modified metallothionein can be produced in prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, mammalian cells, and plant cells. can do. For example, when expressed in a microorganism such as Escherichia coli, an expression vector is prepared by recombining a polynucleotide with an expression vector having an origin, promoter, ribosome binding site, DNA cloning site, terminator, etc. that can replicate in the microorganism, If a host cell is transformed with this expression vector and this transformant is cultured, the desired modified metallothionein can be mass-produced from the culture. Examples of the expression vector for E. coli include pUC system, pBluescript II, pET expression system, and pGEX expression system. Further, when expressing a polypeptide in a eukaryotic cell, a polynucleotide is inserted into an expression vector for a eukaryotic cell having a promoter, a splicing region, a poly (A) addition site, etc. The desired modified metallothionein can be obtained from eukaryotic cells transfected with the vector. Examples of expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYES2. As eukaryotic cells, mammalian cultured cells such as human fetal kidney cells HEK293, monkey kidney cells COS7, Chinese hamster ovary cells CHO, or primary cultured cells isolated from human organs can be used. Budding yeast, fission yeast, silkworm cells, Xenopus egg cells and the like can also be used. In order to introduce the expression vector into cells, known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method can be used. Isolation and purification of the polypeptide expressed in the transformed cell can be performed by combining known separation operations. For example, treatment with denaturing agents and surfactants such as urea, ultrasonic treatment, enzyme digestion, salting out and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
[0032]
As described above, the modified metallothionein of the present invention is characterized in that one or more non-Cys residues in the amino acid sequence constituting the wild-type metallothionein are substituted with Cys residues. Any non-Cys residue may be substituted with a Cys residue, provided that the metal binding ability is enhanced as compared to wild-type metallothionein. For example, human metallothionein-II is composed of 61 amino acids, of which 20 residues are Cys. Therefore, as a result of substituting any one or more of the remaining 41 non-Cys residues with Cys residues, polypeptides whose metal binding ability after the substitution is enhanced as compared with wild-type metallothionein are within the scope of the modified metallothionein of the present invention. include.
[0033]
Such a modified metallothionein can be identified by the screening method of the sixth invention.
[0034]
In the method of the sixth invention, first, as a step (a), a metallothionein mutant in which one or more non-Cys residues in the amino acid sequence of metallothionein is substituted with a Cys residue is used as the type of amino acid residue to be substituted. Prepare a plurality by changing. The metallothionein mutant in this case can be prepared by peptide synthesis as described above, or genetic engineering methods. The number of metallothionein mutants to be prepared is selected from those obtained by substituting 1-41 of non-Cys residues with Cys residues in any combination when, for example, wild-type human metallothionein-II is used as a template. It can be 2 or more.
[0035]
Then, as step (b), each metallothionein variant is contacted with one or more metals. The metal can be selected from cadmium, zinc, copper, mercury, silver and the like to which wild type metallothionein is bound.
[0036]
Then, in step (c), from each of the metallothionein mutants, one having enhanced metal binding ability compared to wild-type metallothionein is selected. One of the criteria is to measure the metal atom bound to each metallothionein mutant and select a metallothionein mutant that binds more metal atoms than wild-type metallothionein. The number of bonds of metal atoms can be determined by a method such as measurement of atomic absorption photometry as shown in the examples. Another criterion for selection is the stability of metallothionein against pH changes (changes in pH increase). That is, the metal bonded to metallothionein dissociates at a low pH (pH 2.5 to 3.0). Metallothionein (apometallothionein) that is not bonded to a metal is easily oxidized and unstable under neutral and alkaline conditions. Therefore, it can be determined that a metallothionein mutant that is more stable than wild-type metallothionein with respect to a change in pH is more strongly bound to a metal.
[0037]
In this application, as the modified metallothionein selected by the screening method as described above, the 11th aspartic acid residue (Asp) and the 28th serine residue (SEQ ID NO: 1) showing the amino acid sequence of human metallothionein ( A modified metallothionein in which Ser) is substituted with a cysteine residue (Cys) is provided as a preferred specific example.
[0038]
Hereinafter, the invention of this application will be described in more detail and specifically with reference to Examples and Test Examples, but the invention of this application is not limited to the following examples.
[0039]
【Example】
Example 1: Construction of polynucleotide encoding metallothionein mutant and expression vector thereof Full-length cDNA of human metallothionein-II (SEQ ID NO: 1) and β domain cDNA (amino acids 1-30 of SEQ ID NO: 1) Using the Mutant-Super Express Km kit (Takara Shuzo Co., Ltd.) for plasmids pTIMTI and pTIMTII (Protein Expr. Purif. 21 (1): 243-250, 2001) expressed as fusion proteins with Intein, site-specific Mutation (base substitution to replace No. 11 Asp and No. 28 Ser with Cys) was introduced. The DNA base sequence of each clone was determined to confirm the target base mutation, and plasmid vectors pTIMTID11C / S28C and pTIMTIID11C / S28C were constructed.
Example 2: Expression and purification of metallothionein mutants The plasmid vectors pTIMTID11C / S28C and pTIMTIID11C / S28C constructed in Example 1 were used to transform E. coli ER2566 strain. Using the IMPACT System (New England Biolabs), let the transformed bacteria express the full-length human metallothionein-II or β-domain and Intein fusion protein, purify using an affinity column, and then dithiothreitol (DDT). Was used to cleave Intein and metallothionein. At each stage of expression and purification, the presence of a high expression band was confirmed by SDS-PAGE. 1N hydrochloric acid is added to the eluted fraction after column purification to pH 1.0, centrifuged at 10,000 rpm for 5 minutes at 4 ° C, and the supernatant is subjected to gel filtration column chromatography (Sephadex G-50 column, 10 ml / hr, 20 mM Tris-HCl solution), each fraction was measured by absorbance at 220 nm, and purified apometallothionein was recovered.
[0040]
In addition, plasmids pTIMTI and pTIMTII were used as controls, and wild type human metallothionein-II and its β domain peptide were expressed and purified in the same manner as described above.
Example 3: Preparation of metal-bonded metallothionein Cadmium chloride and zinc sulfate were added to wild-type and mutant apometallothionein or its β-domain peptide prepared in Example 2 and subjected to gel filtration column chromatography (Sephadex G-50 column). , 10 ml / hr, 20 mM Tris-HCl solution), each fraction is measured at an absorbance of 250 nm when cadmium chloride is added and 220 nm when zinc sulfate is added, and cadmium-bound metallothionein And zinc-binding metallothionein, as well as the respective β domain peptides.
Test Example 1: Measurement of the number of metal bonds
A dilution series (0.01-2.00 μg / ml) of cadmium and zinc was prepared using a standard solution for atomic absorption spectrophotometry (manufactured by Wako) diluted with 0.1N nitric acid, and the metal-bonded metallothionein and β prepared in Example 3 were used. The domain peptide was diluted to about 1.00 μg / ml, and the metal concentration was measured using an atomic absorption spectrophotometer (model SAS7500A: manufactured by Seiko). The measurement wavelength was 229 nm for cadmium and 241 nm for zinc.
[0041]
The results are as shown in Table 1. Compared to wild-type full-length metallothionein and its β-domain peptide, metallothionein mutants and β-domain peptide mutants with two amino acid substitutions were confirmed to bind about one more cadmium and zinc.
[0042]
[Table 1]

[0043]
Test Example 2: Measurement of stability against pH change The metal-bound metallothionein and β domain peptide prepared in Example 3 were diluted to 3 nmol / ml with 20 mM Tris-HCl buffer (pH 7.5), and then pH 2.5 with HCl. To dissociate the metal. Subsequently, Tris solution was added to gradually increase the pH, and the stability of metallothionein and β domain peptide was measured as the change in UV absorption. The absorbance at 250 nm was measured for the cadmium bond and 220 nm for the zinc bond.
[0044]
The results are as shown in FIG. 1 and FIG. FIG. 1 is a pH titration curve of wild-type and mutant cadmium-binding metallothionein and β-domain peptide, and FIG. 2 is a pH titration curve of zinc-binding metallothionein and β-domain peptide. When any metal is bound, compared to wild-type full-length metallothionein and its β-domain peptide, metallothionein mutants and β-domain peptide mutants with two amino acid substitutions rise at a low pH and the gradient is steep. From this, it was confirmed that the pH stability was improved and the metal binding ability was enhanced.
[0045]
【The invention's effect】
As described in detail above, the invention of this application provides a modified metallothionein having further enhanced metal binding ability compared to wild-type metallothionein.
[0046]
[Sequence Listing]
<110> Japan Science and Technology Corporation
<120> A modified metallothionein
<130> NP02127-YS
<140>
<141>
<160> 1
<210> 1
<211> 61
<212> PRT
<213> Homo sapiens
<400> 1
Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala
50 55 60
[Brief description of the drawings]
FIG. 1 is a pH titration curve of wild-type and mutant cadmium-binding metallothionein and β-domain peptide. pTIMTI is a full-length wild type, pTIMTID11C / S28C is a full-length mutant, pTIMTII is a β-domain wild type, and pTIMTIID11C / S28C is a β-domain mutant.
FIG. 2 is a pH titration curve for wild-type and mutant zinc-binding metallothionein and β-domain peptides. pTIMTI is a full-length wild type, pTIMTID11C / S28C is a full-length mutant, pTIMTII is a β-domain wild type, and pTIMTIID11C / S28C is a β-domain mutant.

Claims (10)

A modified metallothionein in which one or more non-cysteine residues in the amino acid sequence of metallothionein are substituted with cysteine residues (Cys), and the binding ability to zinc and cadmium is enhanced as compared to the wild type . The modified metallothionein according to claim 1, wherein the metallothionein is human metallothionein consisting of the amino acid sequence of SEQ ID NO: 1. The modified metallothionein according to claim 2, wherein the 11th aspartic acid residue (Asp) and the 28th serine residue (Ser) in the amino acid sequence of SEQ ID NO: 1 are substituted with a cysteine residue (Cys). A polynucleotide encoding a modified metallothionein in which at least one non-cysteine residue in the amino acid sequence of metallothionein is substituted with a cysteine residue (Cys) and the binding ability to zinc and cadmium is enhanced as compared to the wild type . The polynucleotide of Claim 4 wherein the metallothionein is human metallothionein consisting of the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 encodes a modified metallothionein in which the 11th aspartic acid residue (Asp) and the 28th serine residue (Ser) are substituted with a cysteine residue (Cys). Polynucleotide. A recombinant vector carrying the polynucleotide according to any one of claims 4 to 6. A transformed cell by the recombinant vector of claim 7. A modified metallothionein which is an expression product of the recombinant vector of claim 7 or a product of the transformed cell of claim 8. A method of screening for a modified metallothionein with enhanced metal binding ability, comprising the following steps:
(a) preparing a plurality of metallothionein mutants in which one or more non-cysteine residues in the amino acid sequence of metallothionein are replaced with cysteine residues (Cys) by changing the type of the substituted amino acid residues;
(b) contacting each metallothionein variant with one or more metals selected from the group consisting of cadmium, zinc, copper, mercury, silver ;
(c) selecting a metallothionein mutant that binds more metal atoms than wild-type metallothionein and / or a metallothionein mutant that is more stable against pH change than wild-type metallothionein.
A method for screening a modified metallothionein, comprising:

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